A rifampicin-induced hepatic microsomal enzyme system for the generation of cyclosporine metabolites

Abstract

A drug-induced rabbit hepatic microsomal enzyme system has been developed to produce milligram quantities of cyclosporine metabolites (CMs). Using a rifampicin-induced microsomal preparation in the presence of a NADPH regenerating system, 60% of the cyclosporine (CsA) was converted to CMs in 2 h. The CMs were recovered by solid phase extraction, and separated by gradient high performance liquid chromatography with two Ultrasphere Ocyl (C8) columns connected in tandem. More than 20 CMs were resolved. The quantities of major CMs produced by 45 mg of microsomal proteins were established by comparing peak areas with known concentrations of authentic CM standards. These major CMs included AM1, AM9, AM19, AM4N, AM1c and the aldehydic isomers (AM1cAL plus AM1AL). Other CMs that were not quantified included AM14N, AM4N9, AM1A, AM1c9, and AM1D1. Several CMs remained to be identified. All CMs were detected by radioimmunoassay using a non-specific CsA antiserum. The purity of the CMs were confirmed by fast atomic bombardment mass spectrometry. Similar findings were observed when erythromycin or trolandomycin was used to induce the hepatic microsomal enzymes. The procedure used to generate CMs was simple. With the enzyme fraction derived from one rabbit liver, 90 to 100 mg of CMs can be obtained. In this study, the metabolite patterns of CsA produced by rabbit liver microsomes were shown to resemble those observed for humans. These results indicate the possibility of using rabbit models to predict CsA biotransformation in man. The CMs generated by this enzyme system can be used to acquire information relevant to the situation in man.